, Volume 42, Issue 6, pp 2105–2118 | Cite as

GSK-3β Inhibitor Induces Expression of the TLR4/MyD88/NF-κB Signaling Pathway to Protect Against Renal Ischemia-Reperfusion Injury During Rat Kidney Transplantation

  • Shuai Su
  • Peng Zhang
  • Qilin Zhang
  • Zhikang YinEmail author
Original Article


Ischemia-reperfusion injury (IRI) is an inevitable consequence of kidney transplantation (KT). The aim of our study was to investigate the protective effect of a glycogen synthase kinase 3β (GSK-3β) inhibitor against cold IRI in a rat renal transplantation (RT) model and a rat cold-IRI model through the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor κ-light-chain-enhancer of the activated B cell (NF-κB) signaling pathway. We treated Sprague Dawley (SD) rats in the RT and cold-IRI models with 5 mg/kg and 1 mg/kg, respectively, of the GSK-3β inhibitor 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8). We then measured inflammatory factors, i.e., tumor necrosis factor alpha (TNF-α) and interleukins-1β and IL-6 (IL-1β, IL-6), as well as oxidative stress markers, i.e., superoxide dismutase (SOD) and malondialdehyde (MDA), in serum and kidneys. Renal function tests and pathological examinations were performed at 0, 1, 2, 3, and 7 days after RT or cold IRI. We measured expression of TLR4, MyD88, inhibitor of NF-κB kinase (IκB), phosphorylated IκB (p-IκB), NF-κB p65, p-p65, GSK-3β, and phosphorylated GSK-3β (p-GSK-3β) by Western blot and immunohistological staining. After intervention with the GSK-3β inhibitor, renal function was improved; oxidative stress injury was reduced; expression of p-GSK-3β was upregulated; expression of p-IκB, TLR4, MyD88, and p-p65 was downregulated; pathological damage was significantly reduced; and expression of TNF-α, IL-1β, and IL-6 messenger ribonucleic acid (mRNA) was downregulated. These results strongly suggested that GSK-3β might be a key target for the treatment of IRI in KT. The GSK-3β inhibitor inhibited phosphorylation of NF-κB p65 and IκB by inhibiting the TLR/MyD88 pathway, reducing oxidative stress injury and the production of downstream inflammatory factors.


glycogen synthase kinase 3β TLR4/MyD88/NF-κB pathway renal ischemia-reperfusion injury kidney transplantation 


Compliance with Ethical Standards

All animal procedures for this study were performed strictly according to the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Chongqing Medical University, Chongqing, China.


  1. 1.
    Collins, A.J., R.N. Foley, C. Herzog, et al. 2013. US Renal Data System 2012 Annual Data Report. American Journal of Kidney Diseases 61 (A7): e1–e476.Google Scholar
  2. 2.
    Chen, C.C., W.C. Chapman, and D.W. Hanto. 2015. Ischemia-reperfusion injury in kidney transplantation. Frontiers in Bioscience (Elite Edition) 7: 117–134.Google Scholar
  3. 3.
    Foster, M.C., D.E. Weiner, A.G. Bostom, et al. 2017. Filtration markers, cardiovascular disease, mortality, and kidney outcomes in stable kidney transplant recipients: the FAVORIT Trial. American Journal of Transplantation 17 (9): 2390–2399.PubMedPubMedCentralGoogle Scholar
  4. 4.
    Darrington, R.S., V.M. Campa, M.M. Walker, et al. 2012. Distinct expression and activity of GSK-3α and GSK-3β in prostate cancer. International Journal of Cancer 131 (6): E872–E883.PubMedGoogle Scholar
  5. 5.
    Jellestad, L., T. Fink, S. Pradarutti, et al. 2014. Inhibition of glycogen synthase kinase (GSK)-3-beta improves liver microcirculation and hepatocellular function after hemorrhagic shock. European Journal of Pharmacology 724: 175–184.PubMedGoogle Scholar
  6. 6.
    Cao, Q., A. Karthikeyan, S.T. Dheen, C. Kaur, and E.A. Ling. 2017. Production of proinflammatory mediators in activated microglia is synergistically regulated by Notch-1, glycogen synthase kinase (GSK-3β) and NF-κB/p65 signalling. PLoS One 12 (10): e0186764.PubMedPubMedCentralGoogle Scholar
  7. 7.
    O'Neill, S., D. Humphries, G. Tse, et al. 2015. Heat shock protein 90 inhibition abrogates TLR4-mediated NF-κB activity and reduces renal ischemia reperfusion injury. Scientific Reports 7 (5): 12958.Google Scholar
  8. 8.
    Feng, R., D. Zhongping, C. Qiao, et al. 2011. The inhibition of glycogen synthase kinase 3 beta ameliorates liver ischemia reperfusion injury via an IL-10-mediated immune regulatory mechanism. Hepatology. 54 (2): 687–696.Google Scholar
  9. 9.
    Andrade-Oliveira, V., E.F. Campos, A. Goncalves-Primo, et al. 2012. TLR4 mRNA levels as tools to estimate risk for early posttransplantation kidney graft dysfunction. Transplantation 94: 589–595.PubMedGoogle Scholar
  10. 10.
    Marsh, B.J., R.L. Williams-Karnesky, and M.P. Stenzel-Poore. 2009. Toll-like receptor signaling in endogenous neuroprotection and stroke. Neuroscience. 158: 1007–1020.PubMedGoogle Scholar
  11. 11.
    Chamorro, A., A. Meisel, A.M. Planas, X. Urra, D. van de Beek, and R. Veltkamp. 2012. The immunology of acute stroke. Nature Reviews. Neurology 8: 401–410.PubMedGoogle Scholar
  12. 12.
    Zhang, W.B., H.Y. Zhang, Q. Zhang, et al. 2017. Glutamine ameliorates lipopolysaccharide-induced cardiac dysfunction by regulating the toll-like receptor 4/mitogen-activated protein kinase/nuclear factor-kB signaling pathway. Experimental and Therapeutic Medicine 14 (6): 5825–5832.PubMedPubMedCentralGoogle Scholar
  13. 13.
    Ratilal, B.O., J.P. Rocha, A.M. Fernandes, et al. 2014. TDZD-8 pre-treatment in transient middle cerebral artery occlusion. Biomedicine and Aging Pathology 4 (4): 361–367.Google Scholar
  14. 14.
    Spanjol, J., T. Celic, T. Jakljevic, A. Ivancic, and D. Markic. 2011. Surgical technique in the rat model of kidney transplantation. Collegium Antropologicum 35: 87–90.PubMedGoogle Scholar
  15. 15.
    Lobb, I., J. Jiang, D. Lian, et al. 2017. Hydrogen sulfide protects renal grafts against prolonged cold ischemia-reperfusion injury via specific mitochondrial actions. American Journal of Transplantation 17 (2): 341–352.PubMedGoogle Scholar
  16. 16.
    Ponticelli, C. 2014. Ischaemia-reperfusion injury: a major protagonist in kidney transplantation. Nephrology, Dialysis, Transplantation 29 (6): 1134–1140.PubMedGoogle Scholar
  17. 17.
    Cavaillé-Coll, M., S. Bala, E. Velidedeoglu, et al. 2013. Summary of FDA workshop on ischemia reperfusion injury in kidney transplantation. American Journal of Transplantation 13 (5): 1134–1148.PubMedGoogle Scholar
  18. 18.
    Guo, F., T. Jiang, W. Song, et al. 2015. Electroacupuncture attenuates cerebral ischemia-reperfusion injury in diabetic mice through adiponectin receptor 1-mediated phosphorylation of GSK-3β. Molecular Neurobiology 51 (2): 685–695.PubMedGoogle Scholar
  19. 19.
    Tantray, M.A., I. Khan, H. Hamid, et al. 2016. Synthesis of novel oxazolo [4,5-b]pyridine-2-one based 1,2,3-triazoles as glycogen synthase kinase-3β inhibitors with anti-inflammatory potential. Chemical Biology & Drug Design 87 (6): 918–926.Google Scholar
  20. 20.
    Ren, F., Z. Duan, Q. Cheng, et al. 2011. Inhibition of glycogen synthase kinase 3 beta ameliorates liver ischemia reperfusion injury by way of an interleukin-10-mediated immune regulatory mechanism. Hepatology 54 (2): 687–696.PubMedPubMedCentralGoogle Scholar
  21. 21.
    He, M., Y. Zhang, F. Xie, X. Dou, M. Han, and H. Zhang. 2018. Role of PI3K/Akt/NF-κB and GSK-3β pathways in the rat model of cardiopulmonary bypass-related lung injury. Biomedicine & Pharmacotherapy 106: 747–754.Google Scholar
  22. 22.
    Guo, Y., J. Zhang, X. Lai, M. Chen, and Y. Guo. 2018. Tim-3 exacerbates kidney ischaemia/reperfusion injury through the TLR-4/NF-κB signalling pathway and an NLR-C4 inflammasome activation. Clinical and Experimental Immunology 193 (1): 113–129.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Kim, E., H.C. Kim, S. Lee, et al. 2017. Dexmedetomidine confers neuroprotection against transient global cerebral ischemia/reperfusion injury in rats by inhibiting inflammation through inactivation of the TLR-4/NF-κB pathway. Neuroscience Letters 649: 20–27.PubMedGoogle Scholar
  24. 24.
    Feng, D., Y. Wang, Y. Liu, et al. 2018. DC-SIGN reacts with TLR-4 and regulates inflammatory cytokine expression via NF-κB activation in renal tubular epithelial cells during acute renal injury. Clinical and Experimental Immunology 191 (1): 107–115.PubMedGoogle Scholar
  25. 25.
    Wu, Huiling, Gang Chen, R. Kate, et al. 2007. TLR4 activation mediates kidney ischemia/reperfusion injury. The Journal of Clinical Investigation 117 (10): 2847–2859.PubMedPubMedCentralGoogle Scholar
  26. 26.
    Damman, J., et al. 2011. Crosstalk between complement and Toll-like receptor activation in relation to donor brain death and renal ischemia-reperfusion injury. American Journal of Transplantation 11: 660–669.PubMedGoogle Scholar
  27. 27.
    Zhang, J., P. Yu, M. Chen, Q. Peng, Z. Wang, and N. Dong. 2017. Remote ischaemic preconditioning and sevoflurane postconditioning synergistically protect rats from myocardial injury induced by ischemia and reperfusion partly via inhibition TLR4/MyD88/NF-kappaB signaling pathway. Cellular Physiology and Biochemistry 41: 22–32.PubMedGoogle Scholar
  28. 28.
    Dugo, L., M. Abdelrahman, O. Murch, E. Mazzon, S. Cuzzocrea, and C. Thiemermann. 2006. Glycogen synthase kinase-3β inhibitors protect against the organ injury and dysfunction caused by hemorrhage and resuscitation. Shock 25 (5): 485–491.PubMedGoogle Scholar
  29. 29.
    Zhang, F., C.J. Phiel, L. Spece, N. Gurvich, and P.S. Klein. 2003. Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to lithium. Evidence for autoregulation of GSK-3. The Journal of Biological Chemistry 278: 33067–33077.PubMedGoogle Scholar
  30. 30.
    Ko, R., and S.Y. Lee. 2016. Glycogen synthase kinase 3β in Toll-like receptor signaling. BMB Reports 49 (6): 305–310.PubMedPubMedCentralGoogle Scholar
  31. 31.
    Marko, L., E. Vigolo, and C. Hinze. 2016. Tubular epithelial NF-ΚB activity regulates ischemic AKI. Journal of the American Society of Nephrology 27 (9): 2658–2669.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Cortes-Vieyra, R., A. Bravo-Patino, J.J. Valdez-Alarcon, M.C. Juarez, B.B. Finlay, and V.M. Baizabal-Aguirre. 2012. Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens. Journal of Inflammation 9 (1): 23–31.PubMedGoogle Scholar
  33. 33.
    Schwabe, R.F., and D.A. Brenner. 2002. Role of glycogen synthase kinase-3 in TNF-a-induced NF-κB activation and apoptosis in hepatocytes. American Journal of Physiology. Gastrointestinal and Liver Physiology 283: 204–211.Google Scholar
  34. 34.
    Xu, J., P. Xu, Z. Li, L. Xiao, and Z. Yang. 2013. The role of glycogen synthase kinase-3β in glioma cell apoptosis induced by remifentanil. Cellular & Molecular Biology Letters 18 (4): 494–506.Google Scholar
  35. 35.
    Kimura, T., Y. Isaka, and T. Yoshimori. 2017. Autophagy and kidney inflammation. Autophagy 13 (6): 997–1003.PubMedPubMedCentralGoogle Scholar
  36. 36.
    Schachtner, T., M. Stein, A. Sefrin, N. Babel, and P. Reinke. 2014. Inflammatory activation and recovering BKV-specific immunity correlate with self-limited BKV replication after renal transplantation. Transplant International 27 (3): 290–301.PubMedGoogle Scholar
  37. 37.
    Obligado, S.H., O. Ibraghimov-Beskrovnaya, A. Zuk, L. Meijer, and P.J. Nelson. 2008. CDK/GSK-3 inhibitors as therapeutic agents for parenchymal renal diseases. Kidney International 73 (6): 684–690.PubMedGoogle Scholar
  38. 38.
    Chhabra, M., T.M. Conlon, K. Saeb-Parsy, and G.J. Pettigrew. 2013. BAFF and associated TNF superfamily members in renal transplantation: an end to BLySful ignorance. Transplantation 96 (10): 853–859.PubMedGoogle Scholar
  39. 39.
    Li, Y., D.D. Zhong, L. Lei, Y.L. Jia, H. Zhou, and B.X. Yang. 2015. Propofol prevents renal ischemia-reperfusion injury via inhibiting the oxidative stress pathways. Cellular Physiology and Biochemistry 37 (1): 14–26.PubMedGoogle Scholar
  40. 40.
    Zhao, W., X. Gan, G. Su, et al. 2014. The interaction between oxidative stress and mast cell activation plays a role in acute lung injuries induced by intestinal ischemia-reperfusion. The Journal of Surgical Research 187 (2): 542–552.PubMedGoogle Scholar
  41. 41.
    Qiao, X., R.S. Li, H. Li, et al. 2013. Intermedin protects against renal ischemia-reperfusion injury by inhibition of oxidative stress. American Journal of Physiology. Renal Physiology 304 (1): F112–F119.PubMedGoogle Scholar
  42. 42.
    Jha, J.C., C. Banal, B.S. Chow, M.E. Cooper, and K. Jandeleit-Dahm. 2016. Diabetes and kidney disease: role of oxidative stress. Antioxidants & Redox Signaling 25 (12): 657–684.Google Scholar
  43. 43.
    Li, X.D., G.F. Sun, W.B. Zhu, and Y.H. Wang. 2015. Effects of high intensity exhaustive exercise on SOD, MDA, and NO levels in rats with knee osteoarthritis. Genetics and Molecular Research 14 (4): 12367–12376.PubMedGoogle Scholar
  44. 44.
    Abd-Elsameea, A.A., A.A. Moustaf, and A.M. Mohamed. 2014. Modulation of the oxidative stress by metformin in the cerebrum of rats exposed to global cerebral ischemia and ischemia/reperfusion. European Review for Medical and Pharmacological Sciences 18 (16): 2387–2392.PubMedGoogle Scholar
  45. 45.
    Hsu, S.P., C.C. Chen, and C.T. Chien. 2016. Pretreatment of sialic acid efficiently prevents lipopolysaccharide-induced acute renal failure and suppresses tlr4/gp91-mediated apoptotic signaling. Kidney & Blood Pressure Research 41: 267–277.Google Scholar
  46. 46.
    Jung, M., A. Sola, J. Hughes, et al. 2012. Infusion of IL-10-expressing cells protects against renal ischemia through induction of lipocalin-2. Kidney International 81: 969–982.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Shuai Su
    • 1
  • Peng Zhang
    • 2
  • Qilin Zhang
    • 1
  • Zhikang Yin
    • 1
    Email author
  1. 1.Department of UrologyThe First Affiliated Hospital of Chongqing Medical UniversityChongqingChina
  2. 2.Department of Forensic MedicineHainan Medical UniversityHaikouChina

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